Spermatogonial stem cell regulation and spermatogenesis
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Phil. Trans. R. Soc. B (2010) 365, 1663–1678
doi:10.1098/rstb.2010.0026
Review
Spermatogonial stem cell regulation
and spermatogenesis
Bart T. Phillips†, Kathrin Gassei† and Kyle E. Orwig*
Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute,
University of Pittsburgh School of Medicine, 204 Craft Avenue, Pittsburgh, PA, USA
This article will provide an updated review of spermatogonial stem cells and their role in maintain-
ing the spermatogenic lineage. Experimental tools used to study spermatogonial stem cells (SSCs)
will be described, along with research using these tools to enhance our understanding of stem cell
biology and spermatogenesis. Increased knowledge about the biology of SSCs improves our capacity
to manipulate these cells for practical application. The chapter concludes with a discussion of future
directions for fundamental investigation and practical applications of SSCs.
Keywords: spermatogonial stem cells; spermatogenesis; fertility
1. INTRODUCTION Ying et al. 2001). During the formation of the
Spermatogonial stem cells (SSCs) are at the foun- allantois, the PGCs are passively swept out of the
dation of spermatogenesis and male fertility. Similar embryo before they start migrating via the hindgut to
to other tissue-specific stem cells, SSCs are rare, repre- arrive at the indifferent gonad between 8.5 and
senting only 0.03 per cent of all germ cells in rodent 12.5 dpc in mice. PGCs replicate during the migratory
testes (Tegelenbosch & de Rooij 1993). This is phase and approximately 3000 PGCs colonize the
because SSCs are heavily outnumbered by the differ- genital ridges (Bendel-Stenzel et al. 1998). In the
entiating spermatogonia, spermatocytes, spermatids male gonad at about 13.5 dpc, PGCs give rise to gono-
and sperm that they produce (detailed below). SSCs cytes, which become enclosed in testicular cords
are defined like all other stem cells, by their ability to formed by Sertoli precursor cells and peritubular
balance self-renewing divisions and differentiating div- myoid cells. Gonocyte is a general term that can be
isions. This balance maintains the stem cell pool and subcategorized into mitotic (M)-prospermatogonia,
meets the proliferative demand of the testis to produce T1-prospermatogonia and T2-propsermatogonia
millions of sperm each day. Studies of SSCs are com- (McCarrey 1993). M-prospermatogonia are located
plicated because these cells are few in number and no in the centre of the testicular cords, away from the
unique identifying characteristics have been reported basal membrane and continue proliferating until
to date. We will review experimental tools used to about 16.5 dpc of mouse development when they
study SSCs and summarize current knowledge about become T1-prospermatogonia and enter G0 mitotic
the characteristics and regulation of these adult tissue arrest (McLaren 2003; Tohonen et al. 2003). Gono-
stem cells. We will focus primarily on rodent models, cytes resume proliferation during the first week after
which have generated the majority of data about birth (marking their transition to T2-prospermatogonia),
SSCs and the spermatogenic lineage. concomitant with migration to the seminiferous
tubules basement membrane (Clermont & Perey
1957). T2-prospermatogonia that colonize the base-
2. ORIGIN OF THE SPERMATOGONIAL STEM ment membrane give rise to the first round of
CELL POOL spermatogenesis as well as establish the initial pool of
SSCs arise from gonocytes in the postnatal testis, SSCs that maintain spermatogenesis throughout post-
which arise from primordial germ cells (PGCs) pubertal life (Kluin & de Rooij 1981; McCarrey 1993;
during foetal development. PGCs are a transient cell Yoshida et al. 2006).
population that is first observed as a small cluster of
alkaline phosphatase-positive cells in the epiblast
stage embryo at about 7–7.25 days post coitum (dpc). 3. THE SPERMATOGENIC CYCLE
PGC specification is dependent on the expression of Spermatogenic lineage development is a complex pro-
BMP4 and BMP8b from the extraembryonic ecto- cess, but occurs in an orderly manner, referred to as
derm (Ginsburg et al. 1990; Lawson et al. 1999; the spermatogenic cycle (Clermont 1972), which is
divided in a species-specific number of stages or cell
* Author for correspondence (orwigke@upmc.edu). associations (i.e. 12 stages in the mouse (Oakberg
†
These authors contributed equally to this study. 1956a,b) and 14 stages in the rat (Leblond &
One contribution of 17 to a Theme Issue ‘The biology and Clermont 1952a,b)). This synchronized spermato-
regulation of spermatogenesis’. genic development may be facilitated by incomplete
1663 This journal is # 2010 The Royal Society
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1664 B. T. Phillips et al. Review. Spermatogonial stem cells
cytokinesis during mitotic divisions that lead to main- division, see green clone in figure 1, stages IX, X
tenance of cytoplasmic bridges among germ cells. and XI) or (ii) remains connected by an intercellular
Proteins and messenger RNAs are exchanged via the cytoplasmic bridge and produces a chain of four Aal
cytoplasmic bridges and may help in coordinating spermatogonia at the next division (differentiating div-
the synchronized development of germ cell clones ision, see red clone in figure 1, stages IX and X).
(Braun et al. 1989). Each stage is characterized by a Further cell divisions lead to the formation of chains
combination of the types of spermatogonia, spermato- of 8, 16 and sometimes 32 Aal spermatogonia (see
cytes and spermatids that synchronously proceed red clone in stages XII and I and blue clone in stage
through the spermatogenic process (figure 1). For VII, figure 1). Chains of 4–16 Aal are generally con-
example, the basement membrane of a stage V semini- sidered committed to the differentiation process.
ferous tubule depicted in figure 1 is mostly filled with Thus, the stem cell pool includes As and at least
preleptotene primary spermatocytes (blue cells of a some Apr spermatogonia. Some have argued that stem
large clone). By stage VI, these spermatocytes migrate cell potential may extend to larger clones (e.g. Aal4 or
off the basement membrane and will be replaced by beyond; (Yoshida et al. 2007a; Morimoto et al.
spermatogonia. Thus, stage V can be distinguished 2009)), but this is difficult to confirm experimentally.
from stage VI by the presence or absence of spermato- Note that while each clone can be observed in histo-
cytes on the basement membrane. The duration of logical sections as well as in whole-mount
each stage is precisely timed, and the complete sper- preparations of seminiferous tubules, clone size can
matogenic cycle was determined to be around 8.6 only be observed in the whole-mount preparations
days in the mouse (Oakberg 1956b), and 12.8 days (figure 1).
in the rat (Hilscher et al. 1969). One complete cycle As, Apr and smaller chains of four Aal spermatogo-
(12 stages) of the mouse seminiferous epithelium is nia are evenly distributed along the seminiferous
depicted in figure 1. epithelium (Huckins 1971a,b; Tegelenbosch & de
Rooij 1993). Larger chains of Aal (8, 16 and 32)
become differentiating A1 spermatogonia between
4. SPERMATOGENIC LINEAGE DEVELOPMENT stages IV and VIII of the seminiferous epithelium
In order to understand the regulation of spermato- (there is no cell division at this transition, see blue
gonial stem cells, it is important to understand them clone in figure 1, stages VII and VIII) and these give
in the context of the spermatogenic lineage that they rise to A2 spermatogonia at stage IX (see blue clone
produce. Spermatogonia are primitive diploid germ in figure 1, stage IX). Thus, in contrast to undifferen-
cells, located on the basement membrane of the semi- tiated spermatogonia, differentiating spermatogonia
niferous tubules. Three types of spermatogonia were (A1, A2, A3, A4, intermediate and B) divide in a syn-
initially described based on their nuclear morphology chronized manner and are found at specific stages of
(Roosen-Runge & Giesel 1950; Clermont & Leblond the seminiferous epithelium (for detailed description
1953; Monesi 1962). Type A spermatogonia were con- see Oakberg 1971). B spermatogonia give rise to
sidered the most primitive because heterochromatin is primary spermatocytes that progress into meiosis.
absent from the nucleus, a general characteristic of Two meiotic divisions lead to the formation of second-
undifferentiated cells. The nuclei of intermediate ary spermatocytes and haploid spermatids
type spermatogonia contain a small amount of respectively, which undergo 16 steps of morphological
heterochromatin and type B spermatogonia contain a changes to finally become spermatozoa ready to be
large amount of heterochromatin, indicating a more released from the seminiferous epithelium (Oakberg
differentiated state. 1956a).
Histological staining of whole-mount preparations An alternative to the As model of SSC self-renewal
of seminiferous tubules provided additional level of described above is the A0/A1 model (Clermont &
detail about spermatogonial morphometry compared Bustos-Obregon 1968; Dym & Clermont 1970;
with tissue sections alone and broadened the knowl- Clermont & Hermo 1975). This model is very similar
edge of the spermatogonial cell types in the testis. to the Adark and Apale model that has been used to
To facilitate the following discussion, figure 1 depicts describe stem cell activity in non-human primates
one compete cycle of the mouse seminiferous epi- (Clermont & Leblond 1959; Clermont & Antar
thelium and represents whole-mount perspective as 1973). Briefly, A0 spermatogonia were observed as
well as corresponding cross-section and longitudinal singles or pairs of cells that were present at all stage
section perspectives. Figure 1 traces the development of the seminiferous epithelium. Mitotic figures were
of three putative clones (green, red and blue) through rarely observed in these cells, so they were considered
one cycle of the seminiferous epithelium. Based on ‘reserve stem cells’ not contributing to steady-state
whole-mount examination of seminiferous tubules, spermatogenesis. These reserve stem cells are only
Huckins & Oakberg (Huckins 1971c; Oakberg 1971) activated when spermatogenesis is destroyed by toxic
reported that undifferentiated type A spermatogonia insult (i.e. radiation). The ‘active stem cell’ pool is
can be subdivided into Asingle (As), Apaired (Apr) and comprised of A1 – A4 spermatogonia. When A4 sper-
Aaligned (Aal) spermatogonia, which differ only in matogonia divide, they give rise either to new A1
their topographical arrangement on the seminiferous spermatogonia (self-renewal) or to intermediate sper-
tubule basement membrane. When an As (see green matogonia (differentiation). While there continues to
clone in figure 1, stage VII) spermatogonium divides, be vigorous debate about the merits of the As versus
it produces an Apr that either (i) completes cytokinesis the A0/A1 models, the As model is currently favoured
to produce two new As spermatogonia (self-renewing by most investigators in the field and will be the basis
Phil. Trans. R. Soc. B (2010)
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Review. Spermatogonial stem cells B. T. Phillips et al. 1665
cross-section whole-mount
VII VIII
IX
X
longitudinal section XI
XII
I II
III
VI IV
V
Figure 1. Mouse spermatogenic clone development by stage. The mouse spermatogenic cycle contains twelve stages (I– XII).
Each stage is temporally unique, and the stages in the diagram represent the relative time each stage lasts in the mouse. Each
stage in the diagram is shown in cross-sectional, longitudinal and whole-mount perspectives (labelled in stage VII). Three
putative spermatogonial clones are highlighted in blue, red and green. The dotted lines in the whole-mount perspective indi-
cate the planes of the cross section and longitudinal section views. For example, in stage VII, the red cell is in the vertical line
and therefore appears in the cross-sectional view. A green cell is in the horizontal line, so is observed in the longitudinal section
view. The development of three putative clones (blue, red and green) through one cycle of the seminiferous epithelium is
shown. Stage VII: Aal-16 (blue); Apair (red); Asingle (green); stage VIII: A1 (clone of 16) (blue); Apair (red); Asingle (green);
stage IX: A2 (clone of 32) (blue); Apair (red); Asingle (green); stage X: A2 (clone of 32) (blue); Aal-4 (red); Apair (green);
stage XI: A3 (clone of 64) (blue); Aal-4 (red); Asingle (x2) (green); stage XII: A3 (clone of 64) (blue); Aal-4 (red); Asingle
(x2) (green); stage I: A4 (clone of 128) (blue); Aal-8 (red); Asingle and Apair (green); stage II: intermediate spermatogonia
(clone of 256) (blue); Aal-8 (red); Asingle and Apair (green); stage III: intermediate spermatogonia (clone of 256) (blue);
Aal-8 (red); Asingle and Apair (green); stage IV: Type B Spermatagonia (clone of 512) (blue); Aal-8 (red); Asingle and Apair
(green); stage V: Type B Spermatagonia (clone of 512) (blue); Aal-8 (red); Asingle and Apair (green); stage VI: primary sperma-
tocytes (lifting off the basement membrane) (blue); Aal-8 (red); Asingle and Apair (green).
for further discussion of spermatogonial self-renewal 6. SPERMATOGONIAL STEM CELL
in this review. TRANSPLANTATION
A technique for transplanting SSCs was first described
by Brinster and colleagues in 1994 (Brinster & Avar-
5. EXPERIMENTAL TOOLS FOR STUDYING bock 1994; Brinster & Zimmermann 1994). Briefly,
SPERMATOGONIAL STEM CELLS germ cells are isolated from the testes of donor animals
As discussed above, experimental investigation of and transplanted into the testicular seminiferous
SSCs is complicated because these cells are rare and tubules of infertile recipients, where they produce
are difficult to distinguish from the differentiating pro- normal colonies of spermatogenesis and functional
geny that they produce. Whole-mount analyses of sperm (figure 2). Infertility of recipients is because of
seminiferous tubules help in distinguishing As from genetic mutation (i.e. W mutant mice, (Ogawa et al.
Apr and Aal spermatogonia, but there is continuing 2000)) or induced experimentally (e.g. busulphan
debate about whether the stem cell pool is restricted treatment (Brinster & Zimmermann 1994)). In mice,
to As or might be expanded to include Apr and some these studies are facilitated by the availability of trans-
Aal (Nakagawa et al. 2007; Yoshida et al. 2007a). genic donors (e.g. lacZ and GFP) with germ cells that
Thus, the only way to definitively identify an SSC is can be readily identified in the testes of non-transgenic
by observing its biological capacity to produce and recipients. By definition, only a stem cell can produce
maintain spermatogenesis in a transplant paradigm. and maintain a colony of spermatogenesis and
Phil. Trans. R. Soc. B (2010)
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1666 B. T. Phillips et al. Review. Spermatogonial stem cells
Figure 2. Spermatogonial stem cell (SSC) transplant assay. The functional analysis of SSCs is a retrospective assay of sperma-
togenic function. In this example, cells are isolated from a lacZ donor mouse testis and digested to produce a single cell
suspension. Cells can then be maintained in culture or injected into the testes of an infertile recipient mouse. Recipient
testes are typically analysed two to three months after transplantation for donor spermatogenesis (blue colonies in this
example). A typical recipient testis is shown with blue colonies of donor-derived spermatogenesis (scale bar, 2 mm).
each colony arises from the clonogenic proliferation that recognizes a cell surface antigen. Markerþ and
and differentiation of a single SSC (Dobrinski et al. marker2 cells are fractionated by FACS and each frac-
1999; Zhang et al. 2003; Kanatsu-Shinohara et al. tion is transplanted into the seminiferous tubules of
2006). Therefore, the SSC transplantation technique infertile recipient mice to determine the relative stem
provides a quantitative functional assay to characterize cell activity. The first application of this approach for
stem cell activity in any donor cell population. SSC characterizing SSCs was reported by Shinohara and
transplantation remains the gold standard method co-workers, who demonstrated that SSCs specifically
for identifying SSCs, but this approach can be bind to laminin-coated plates. The laminin-binding
technically challenging. In addition, SSC transplan- cells were enriched for b1-integrin, making this sur-
tation is a retrospective assay with an inherent two face molecule a candidate for enriching SSCs
to three months timeframe between transplant and (Shinohara et al. 1999). Subsequent transplantation
analysis. To accelerate investigations of SSCs, of magnetic-activated cell-sorted (MACS) and
Nagano and co-workers recently suggested that the FACS-sorted testis fractions indicated that SSCs
SSC culture system (described below) may provide a express b1-integrin and a6-integrin, but are negative
shorter term, in vitro assay for SSCs (Yeh et al. for av-integrin and the c-KIT receptor tyrosine
2007). However, culture does not assess regenerative kinase (Shinohara et al. 1999, 2000). Based on several
activity. similar studies, mouse SSCs can now be described by
the cell surface phenotype, a6-Integrin (CD49f)þ, b1-
Integrin (CD29)þ, THY-1 (CD90)þ, CD9þ,
7. DISSECTING THE MOLECULAR PHENOTYPE GFRa1þ, CDH1þ, av-Integrin (CD51)2, c-KIT
OF SPERMATOGONIAL STEM CELLS (CD117)2, major histocompatibility complex class I
Fluorescence-activated cell sorting (FACS), combined (MHC-I)2, CD452 (Shinohara et al. 1999, 2000;
with SSC transplantation is a powerful tool that has Kubota et al. 2003; Kanatsu-Shinohara et al. 2004b;
enabled investigators to systematically characterize Buageaw et al. 2005; Fujita et al. 2005; Hofmann
cell surface molecules of SSCs. This experimental et al. 2005b; Lo et al. 2005; Tokuda et al. 2007,
approach is patterned after similar studies to charac- table 1). Using combinations of positive and negative
terize and enrich haematopoietic stem cells markers, it is now possible to achieve significant
(Spangrude 1989; Smith et al. 1991; Osawa et al. enrichment (100- to 200-fold) of mouse SSCs
1996). Briefly, a heterogeneous testis cell suspension (Shinohara et al. 2000; Kubota et al. 2003). However,
is stained with a fluorescent-conjugated antibody it should be noted that none of these markers are
Phil. Trans. R. Soc. B (2010)
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Review. Spermatogonial stem cells B. T. Phillips et al. 1667
Table 1. Germ cell markers in the rodent testis.
germ cell type
germ cell
markers in the experimental transplantable A1–
rodent testis evidence SSC?a As Apr Aal 4 In B Spc RS ES references
c-kit Mu, RT–PCR, no X X X X X X Manova et al.
ISH, IHC, Tr (1990), Yoshinaga
et al. (1991),
Schrans-Stassen
et al. (1999) and
Shinohara et al.
(2000)
GCNA1 WB, IHC not tested X X X X X X X X Enders & May
(1994)
VASA (MvH) ISH, WB, IHC, not tested X X X X X X X X Fujiwara et al.
KO (1994), Tanaka
et al. (2000) and
Toyooka et al.
(2000)
EE2 antigen WB, IHC, not tested X X X X X X X Koshimizu et al.
(1995)
DAZL RT–PCR, NB, not tested X X X X X X X Cooke et al. (1996),
ISH, KO, Niederberger et al.
IHC (1997) and Ruggiu
et al. (1997)
Stra8 RT–PCR, ISH, yes X X X X X X X X X Oulad-Abdelghani
IHC, WM, et al. (1996),
TG, MACS, Giuili et al. (2002)
Tr and Antonangeli
et al. (2009)
a6-integrin FC, MACS, Tr yes X X X X X X X Shinohara et al.
(CD49f) (1999, 2000)
b1-integrin FC, MACS, Tr yes X X X X X X Shinohara et al.
(CD29) (1999) and
Kanatsu-
Shinohara et al.
(2008)
Epcam IHC, MACS not tested X X X X X X Anderson et al.
(1999), van der
Wee et al. (2001)
and Tokuda et al.
(2007)
Pou5f1 (Oct4) IHC, WM, TG, yes X X X X Pesce et al. (1998),
FC, Tr, ISH Yoshimizu et al.
(1999), Ohbo
et al. (2003) and
Ohmura et al.
(2004)
GFR-a1 TG, ISH, IHC, yes X X X Meng et al. (2000),
MACS, TR, Dettin et al.
WM (2003), Buageaw
et al. (2005) and
Grisanti et al.
(2009)
CD24 FC not tested X X X Kubota et al. (2003)
Thy1 (CD90) FC, TR yes X X X Kubota et al. (2003)
Nanos2 ISH, KO, WB, yes X X Tsuda et al. (2003),
RT– PCR, Suzuki et al.
Tg, IHC, (2007, 2009) and
TR, WM Sada et al. (2009)
Nanos3 NB, ISH, KO, not tested X X X Tsuda et al. (2003),
WB, RT– Suzuki et al.
PCR, Tg, (2007, 2009) and
IHC, WM Lolicato et al.
(2008)
(Continued.)
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1668 B. T. Phillips et al. Review. Spermatogonial stem cells
Table 1. (Continued.)
germ cell type
germ cell
markers in the experimental transplantable A1 –
rodent testis evidence SSC?a As Apr Aal 4 In B Spc RS ES references
CD9 FC, IHC, yes X X X X X X Kanatsu-Shinohara
MACS, Tr et al. (2004b)
EGR3 IVC, IHC not tested X X Hamra et al. (2004)
Ngn3 ISH, TG, WM, yes (approx. X X X X Yoshida et al. (2004,
IHC, Tr 10% of 2006) and Raverot
SSCs) et al. (2005)
PLZF Mu, KO, Tr, yes X X X Buaas et al. (2004),
WM, FC, Costoya et al.
ISH, IHC (2004) and
Grisanti et al.
(2009)
RBM RT–PCR, IHC not tested X X X X Jarvis et al. (2005)
Sox-3 KO, IHC not tested X X X Raverot et al. (2005)
TAF4B KO, IHC not tested X X X X X X X X Falender et al.
(2005)
Bcl6b siRNA not tested X X X Oatley et al. (2006)
Numb NB, WB, IHC not tested X X X X X X X Corallini et al.
(2006)
Lrp4 WB, IHC not tested X X X X X X X X X Yamaguchi et al.
(2006)
Ret IHC, MACS, no X X X Ebata et al. (2005)
Tr and Naughton
et al. (2006)
Sohlh1 KO, RT–PCR, not tested X X X X X Ballow et al. (2006a)
IHC,
Sohlh2 RT–PCR, IHC not tested X X X Ballow et al. (2006b)
CDH1 IHC, WM, yes X X X Tokuda et al. (2007)
(CD324) MACS, Tr
GPR125 TG, FC, IVC, yes X X X Seandel et al. (2007)
Tr
Nucleostemin TG, IHC, FC, yes X X X X X X X Ohmura et al. (2008)
Tr, IVC,
siRNA
UTF1 RT–PCR, IHC not tested X X X van Bragt et al.
(2008)
Lin28 (Tex17) IHC, WM, not tested X X X Zheng et al. (2009)
WB, siRNA
As, A single spermatogonia; Apr, A paired spermatogonia; Aal, A aligned spermatogonia; A1–4, differentiating type A1 to A4
spermatogonia; In, intermediate type spermatogonia; B, type B spermatogonia; Spc, spermatocytes; RS, round spermatids; ES, elongated
spermatids; Mu, mutant mouse; TG, transgenic mouse; KO, Knockout mouse; Tr, germ cell transplantation; IHC,
immunohistochemistry; WM, whole mount immunostaining; FC, flow cytometry (including FACS); IVC, in vitro culture; WB, Western
blot; NB, Northern blot; ISH, in situ hybridization; RT–PCR, reverse transcriptase– PCR; siRNA, in vitro knockdown experiment using
siRNA; MACS, magnetic-activated cell sorting.
a
As determined by the spermatogonial stem cell transplantation assay.
exclusive to SSCs and no marker or combination of faithful expression of lacZ and GFP transgenes
markers has produced a pure population of SSCs. (Yeom et al. 1996; Yoshimizu et al. 1999). The
Also, while FACS and MACS sorting followed by Oct-4 – GFP mouse is a valuable tool that enabled
transplantation are powerful tools for characterizing FACS-based isolation and transplantation of Oct4
the cell surface phenotype of SSCs, this approach expressing germ cells from a heterogeneous testis cell
has limited utility for characterizing cytoplasmic or suspension (Ohbo et al. 2003; Ohmura et al. 2004).
nuclear markers. Stem cell activity was significantly enriched in the
Genetic mouse models in which GFP expression is Oct4 expressing (GFPþ) population compared with
driven by a promoter from a putative SSC gene pro- the Oct4 negative (GFP2) population of mouse
vide an alternative approach for characterizing SSCs. testis cells (Ohmura et al. 2004). Interestingly, gono-
For example, Schöler and co-workers reported that cytes and pre-spermatogonia from neonatal mice
the OCT-4 transcription factor is expressed by gono- with an Oct4 –EGFPþ/c-Kit2 phenotype had a greater
cytes and type A spermatogonia of newborn, pup repopulation capacity than Oct4 – EGFPþ/c-Kitþ cell
and adult mouse testes (Pesce et al. 1998). This fractions (Ohbo et al. 2003). These data suggest that
group subsequently characterized an 18 kb promoter/ there is molecular heterogeneity among pre-spermato-
enhancer fragment of the Oct-4 gene that directed gonia. This observation is consistent with reports
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Review. Spermatogonial stem cells B. T. Phillips et al. 1669
suggesting that some gonocytes/pre-spermatogonia male germ lineage. In this context, a candidate SSC
establish the initial pool of SSCs, while other gono- marker would be expressed by cells located on the
cytes/pre-spermatogonia differentiate to produce the basement membrane of seminiferous tubules and be
first round of spermatogenesis (Kluin & de Rooij co-expressed with confirmed markers of SSCs. This
1981; Yoshida et al. 2006). histochemical approach is most convincing in whole-
Transgenic and conditional knock-in approaches mount preparations of seminiferous tubules in which
were recently used to demonstrate that neurogenin 3 it is possible to correlate marker expression with
(Ngn3) is expressed by the earliest spermatogonia clone size (i.e. As, Apr, Aal). Several established mar-
(Yoshida et al. 2004), including at least 11 per cent kers of stem, progenitor and differentiating
of transplantable SSCs (Nakagawa et al. 2007). The spermatogonia are listed in figure 3. Here we define
fact that Ngn3 was not expressed by all transplantable progenitors as undifferentiated spermatogonia that
stem cells in that study provides additional evidence are committed to differentiate. An example of this
that there may be heterogeneity among SSCs. A con- approach is shown in figure 4 for the putative SSC
ditional knock-in approach was also used to marker, Spalt-like 4 (SALL4). SALL4 is a zinc finger
demonstrate that Nanos2 is expressed by SSCs (Sada transcription factor that is expressed in the inner cell
et al. 2009). Finally, transgenic models suggest that mass of the late blastocyst in a pattern similar to
Stra-8 (stimulated by retinoic acid-8) is expressed by OCT4 and SOX2 (Elling et al. 2006). In vitro,
undifferentiated spermatogonia, including SSCs SALL4 stimulates embryonic stem (ES) cell prolifer-
(Giuili et al. 2002; Guan et al. 2006; Sadate-Ngatchou ation (Sakaki-Yumoto et al. 2006) and maintains
et al. 2008), although the transplant data in the Stra-8 pluripotency by repressing trophectoderm differen-
studies were limited. tiation (Yuri et al. 2009), possibly by binding the
Knock-out models have also been used to demon- Oct-4 proximal promoter (Zhang et al. 2006) and by
strate that specific genes/proteins are required for interacting with NANOG (Wu et al. 2006). Thus,
SSC function. Male mice carrying the luxoid (lu) SALL4 is an important stemness factor and together
mutation are subfertile and show abnormal sperm with OCT-4, SOX2 and NANOG constitutes a tightly
development. Progression of infertility is caused by regulated transcription circuit important for stem cell
gradual loss of SSCs (Buaas et al. 2004). The mutation pluripotency (Lim et al. 2008; Yang et al. 2008). Post-
was shown to affect the Zfp145 locus, which encodes natally, Sall4 expression is restricted to the gonads and
the transcriptional repressor PLZF (promyelocytic is expressed by isolated spermatogonia (Wang et al.
leukaemia zinc-finger). PLZF is expressed during 2001; Sall4 was identified as testis-expressed gene 20
embryogenesis and plays a crucial role during limb (Tex20) in that paper). Co-stained whole-mount semi-
and axial skeletal patterning. Targeted disruption of niferous tubules (figure 4) indicated that SALL4 is
Zfp145 resulted in a testicular phenotype similar to expressed by single, paired and aligned cells on the
that of luxoid mutant mice (Costoya et al. 2004). In seminiferous tubule basement membrane and overlaps
the testis, PLZF expression is restricted to As, Apr with consensus SSC markers, PLZF (figure 4a – c)
and Aal undifferentiated spermatogonia, including and GFRa1 (figure 4d – f ). However, these whole-
SSCs as demonstrated by transplantation experiments mount immunohistochemistry results highlight the
of testicular cells from luxoid or PLZF 2/2 mice that heterogeneity among undifferentiated spermatogonia,
failed to initiate donor-derived spermatogenesis in reci- including As spermatogonia (see figure 4f with
pient mice (Buaas et al. 2004; Costoya et al. 2004). A examples of SALL4þ/GFRa12 and SALL4þ/
possible role for PLZF in spermatogonia could be the GFRa1þ As spermatogonia). GFRa1 appears to have
maintenance of an undifferentiated state (Filipponi the most restricted expression (limited to singles,
et al. 2007), similar to the role suggested for Plzf in pairs and chains of four), while PLZF and SALL4
haematopoietic precursor cells (Reid et al. 1995). are also expressed by larger chains of 8 and 16 Aal
Similar knock-out and over-expression studies spermatogonia.
implicate glial cell line-derived neurotrophic factor Similar observations of molecular heterogeneity
(GDNF) and its receptor GFRa1 in stem cell self- among undifferentiated As, Apr and Aal spermatogonia
renewal (Meng et al. 2000). GDNF signalling has have been reported in several recent studies (Tokuda
since been shown to be required for in vitro expansion et al. 2007; Grisanti et al. 2009; Sada et al. 2009;
of SSCs, and it has been demonstrated that a combi- Suzuki et al. 2009; Zheng et al. 2009). The functional
nation of GDNF and soluble GFRa1 is most significance of this heterogeneity remains to be deter-
favourable for the self-renewal of SSCs in vitro mined. Through the combination of FACS and
(Kubota et al. 2004a,b; see below). Finally, knock-out MACS analyses, transplantation, genetic models and
studies implicate Sox3 in the differentiation of the ear- histochemical approaches, the phenotype of rodent
liest germ cells (Raverot et al. 2005). The latter study SSCs is beginning to emerge. A list of putative SSC
indicated that Ngn3 expression is dependent on SOX3 and undifferentiated spermatogonia markers is pro-
and suggested that SOX3 may act directly or indirectly vided in table 1 along with the experimental evidence
through Ngn3 to regulate spermatogonial differentiation used to characterize each marker.
(Raverot et al. 2005).
In addition to data derived from flow cytometry,
genetic models and transplantation, immunohisto- 8. THE SPERMATOGONIAL STEM CELL NICHE
chemistry in tissue sections or intact seminiferous SSCs reside within a specialized microenvironment
tubules (whole mount) has been widely used to inves- called ‘niche’ that regulates testicular homeostasis by
tigate the expression patterns of various proteins in the balancing SSC self-renewal and differentiation. A
Phil. Trans. R. Soc. B (2010)
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1670 B. T. Phillips et al. Review. Spermatogonial stem cells
stem cell
stem As Oct4 + Sohlh2 +
Gfrα1 + Cdh1 +
Ngn3 +/– Utf1 +
Plzf + Lin28 +
Apair Bcl6b + Sall4 +
undifferentiated
progenitor
Oct4 + Sohlh2 +
Aaligned(4) Gfrα1 + Cdh1 +
Ngn3 + Utf1 +
Plzf + Lin28 +
Bcl6b + Sall4 +
progenitor Aaligned(16) Sohlh1 +
differentiating differentiating
A1
spermatogonia
cKit + Sohlh1 +
Ngn3 +
Figure 3. Genes expressed by stem, progenitor and differentiating spermatogonia. The As, seen at the top of the diagram, is
responsible for self renewal and differentiation. Self-renewal is represented here by the Apair dividing to form two As. Differ-
entiation is indicated by colour change (from dark to light) and the lengthening chain of germ cells. Genes are listed with their
expression at the given stages of spermatogonial development. While stem cell activity is considered to reside in the pool of As
spermatogonia, the tapered triangle on the left indicates that stem cell activity may extend to Apr and some Aal spermatogonia.
stem cell niche is comprised of cells, extracellular (Chiarini-Garcia et al. 2003). Undifferentiated sper-
matrix components, and local soluble factors present matogonia are observed predominantly in tubule
in the vicinity of the stem cell that regulates cell fate. areas adjacent to vasculature (Yoshida et al. 2007b;
The structural basis for the SSC niche in the figure 5a).
mammalian testis is the basal compartment of the The SSC niche mediates endocrine and paracrine
seminiferous tubules that is composed of Sertoli cells signals that regulate self-renewal and differentiation
and peritubular myoid cells (Dadoune 2007) (figure 5b). A key regulator of the SSC niche is
(figure 5). Together, Sertoli and peritubular myoid GDNF which is secreted by Sertoli cells and acts
cells secrete the basement membrane components to through Ret receptor tyrosine kinase and GFRa1
which the SSCs are connected via adhesion molecules co-receptor, which form a receptor complex on the sur-
(Tung et al. 1984). Sertoli cells are polarized columnar face of As, APr and Aal (Meng et al. 2000). Downstream
epithelial cells that support SSCs and differentiating signalling pathways that are activated by GDNF in
germ cells by providing nutrients and mediating exter- undifferentiated spermatogonia are the PI3K/Akt path-
nal signals in order to support spermatogenesis way, members of the Src kinase family and the Ras/
(Griswold 1998). The importance of Sertoli cells for Erk1/2 pathway (Braydich-Stolle et al. 2007; Oatley
germ cell differentiation is demonstrated by the trans- et al. 2007; He et al. 2008). GDNF is thought to act
plantation of normal Sertoli cells into the testis of through these pathways to regulate SSC self-renewal.
infertile mutant recipients with a Sertoli cell defect Targeted disruption of the Ets variant gene 5
and successful initiation of spermatogenesis by recipi- (Etv5) results in defective maintenance of the SSC
ent-derived spermatogonia (Kanatsu-Shinohara et al. pool, whereas spermatogonial differentiation appears
2003b, 2005b). Tight junctions between adjacent to be unaffected by this mutation (Chen et al.
Sertoli cells constitute a protective blood –testis barrier 2005). The transcription factor Etv5 is expressed in
(BTB) that divides the seminiferous epithelium into Sertoli cells and loss of Etv5 appears to impair the
basal and adluminal compartments (figure 5a) and ability of Sertoli cells to support spermatogonia,
plays an important role in the regulation of germ cell possibly by disrupted BTB function as indicated by
differentiation (Cheng & Mruk 2002). The BTB decreased Claudin-5 (CLDN5) levels in mutant
maintains a selective substance flow between luminal mice (Morrow et al. 2009). In Sertoli cells, Etv5 is
fluid, blood plasma and interstitial fluid, thereby creat- upregulated by FGF2 in vitro, which is important
ing an immune-privileged environment for haploid for SSC renewal in culture (Kanatsu-Shinohara et al.
germ cells in the adluminal compartment of the 2003a; Kubota et al. 2004a; Yoon et al. 2009). There-
seminiferous tubules. fore, in addition to a direct effect of FGF2 on SSCs,
Along the length of the tubule, SSCs are thought to an indirect paracrine effect of FGF2 on Sertoli cells
be localized in areas adjacent to the interstitial space appears possible.
Phil. Trans. R. Soc. B (2010)Downloaded from http://rstb.royalsocietypublishing.org/ on November 1, 2015
Review. Spermatogonial stem cells B. T. Phillips et al. 1671
(a) (b) (c)
Apr Apr
As As
As
Aal Aal Aal
Sall4 PLZF merge
(d ) (e) (f)
Aal Aal
As As
Aal
As As
Apr Apr
Apr
Apr
Sall4 Gfrα1 merge
Figure 4. Immunofluorescent co-staining of adult mouse whole-mount seminiferous tubules. (a) SALL4 labels undifferen-
tiated As, Apr and Aal spermatogonia. (b) PLZF labels undifferentiated As, Apr and Aal spermatogonia. (c) Merged picture
from (a,b). SALL4 and PLZF are mostly co-expressed in undifferentiated spermatogonia. (d–f ) Co-staining of SALL4 and
GFRa1 reveals heterogeneity within the population of undifferentiated spermatogonia. GFRa1 expression appears more
restricted than SALL4 or PLZF. Scale bar, 50 mm.
The importance of peritubular myoid cells for sper- testis cell populations (FACS or MACS sorting and/
matogonia maintenance has long been discussed. New or differential attachment and replating) resulted in
data now suggest a role for the peritubular cell product the enrichment of SSCs and the removal of somatic
colony-stimulating factor 1 (CSF1) on SSC mainten- cells that promote germ cell differentiation. Second,
ance (Oatley et al. 2009). Csf1 was found to be development of a serum-free, defined medium facili-
expressed in interstitial Leydig and peritubular myoid tated the discovery of essential growth factors.
cells, whereas the Csf1 receptor (Csf1r) was highly Specifically, GDNF is necessary to maintain and
enriched in THY1þ cell fractions from pre-pubertal expand rodent SSCs in culture (Kanatsu-Shinohara
and adult mouse testis. et al. 2003a; Kubota et al. 2004b). The trophic effects
of GDNF in both mice and rats is enhanced by the
addition of soluble GFRa1 (the receptor for GDNF)
9. SSC CULTURE and FGF2 (Kubota et al. 2004a; Ryu et al. 2005).
SSC culture provides a new approach for investigating Unlike mouse ES cells, the additions of leukaemia
the molecular mechanisms and cell-signalling path- inhibitory factor (LIF) and foetal bovine serum
ways that regulate SSC function. While methods for (FBS) to cultures are superfluous and detrimental,
maintaining and amplifying pluripotent ES and respectively, in SSC cultures (Kubota et al. 2004b).
embryonic germ cells in culture are routine, methods Third, STO or mouse embryonic fibroblast (MEF)
for culturing adult tissue stem cells (including SSCs) feeder cells are usually required. Whereas Shinohara’s
had been more difficult to establish. However, tremen- group has demonstrated that mouse SSCs can also
dous progress culturing mouse and rat SSCs has been be maintained in feeder-free conditions (Kanatsu-
reported during the past 5 – 6 years (Kanatsu- Shinohara et al. 2005a). SSC cultures are usually estab-
Shinohara et al. 2003a; Kubota et al. 2004a,b; lished from mouse pup testes (5–12 days postpartum)
Hamra et al. 2005; Ryu et al. 2005). Rodent SSCs because SSCs are enriched at this stage of develop-
can now be maintained for a very long time (perhaps ment. However, SSC cultures can be established from
indefinitely) with a significant amplification in neonate (Kanatsu-Shinohara et al. 2003a) and adult
numbers. Stem cell activity in these cultures was con- mouse testis cells (Kubota et al. 2004a). Immortalized
firmed by SSC transplantation, as diagrammatically SSC lines have been established by the introduction of
represented in figure 2. The doubling time for mouse a retroviral telomerase gene (Feng et al. 2002) or treat-
SSCs was determined to be 5.6 days (Kubota et al. ment with the SV40 large T-antigen (Hofmann et al.
2004b), while the doubling time for rat SSCs is 3– 4 2005a). Evidence that each of these immortalized cell
days (Hamra et al. 2005) or 11 days (Ryu et al. 2005). lines is spermatogonial-like is based primarily on gen-
Several factors were critical to the establishment of etic or immunocytochemical data, but transplantation
long-term SSC cultures. First, methods to fractionate data are lacking.
Phil. Trans. R. Soc. B (2010)Downloaded from http://rstb.royalsocietypublishing.org/ on November 1, 2015
1672 B. T. Phillips et al. Review. Spermatogonial stem cells
(a) containing on Oct-4-targeted shRNA. The treatment
caused a significant reduction in OCT-4 expression
and reduced colonizing activity in the transplant
assay by sixfold. Thus, through genetic manipulation
adluminal and transplantation of SSC cultures, studies will con-
tinue to unravel regulatory pathways required for
SSC self-renewal and differentiation.
basal
10. FUTURE DIRECTIONS
We have attempted to review the current state of
interstitial knowledge and research in the biology of SSCs,
focused primarily on the rodent model. Many areas
of research are only beginning to be thoroughly inves-
tigated in SSCs, such as the molecular regulation of
(b) myoid cell blood vessel stem cell fate decisions and SSC heterogeneity.
Recent progress characterizing, manipulating and cul-
turing SSCs has opened the door to new experimental
approaches for fundamental investigation and possible
CSF-1 SSC ? practical applications discussed below.
In vitro derivation of haploid gametes (elongated
spermatids or sperm) may help to overcome spermato-
genic barriers in infertile men. Feng et al. (2002)
GDNF testosterone (?) reported the production of spermatocytes and sperma-
tids from a stable mouse SSC line, though the
fertilization potential of these cells was not tested.
testosterone
Haploid male germ cells have also been generated by
Sertoli cell Leydig cell differentiation of ES cells in mice (Toyooka et al.
2003; Geijsen et al. 2004; Nayernia et al. 2006) and
Figure 5. SSC niche. The SSC (dark blue) is diagrammed in humans (Kee et al. 2009). Two studies demonstrated
its physical niche (a) surrounded by Sertoli cells (orange) that in vitro, ESC-derived spermatids were competent
and differentiating germ cells (light blue) within the semini- to fertilize mouse eggs, generating blastocysts (Geijsen
ferous tubule. Niche components outside the tubule itself
2004) and live progeny (Nayernia et al. 2006), respect-
include myoid cells (green), blood vessels (red) and Leydig
cells (yellow). The components of the niche and the some ively. However, because of the epigenetic
factors known to be provided by each are shown in (b). reprogramming that occurs during in vivo germ cell
While some factors are known to act directly on the SSC, development, the epigenetic regulation of in vitro
such as GDNF, others, like testosterone are important for gametogenesis must be carefully assessed before clini-
spermatogenesis but may not act on the SSC. cal applications ensue (Georgiou et al. 2007).
Generation of haploid germ cells from primary SSC
cultures has not yet been reported, but this approach
Stable SSC culture provides a valuable tool for dis- may have epigenetic advantages over ESC-derived
secting mechanisms that regulate SSC renewal and gametes. Furthermore, progress establishing human
differentiation. GDNF is required for SSC renewal SSC cultures will be an important experimental tool
in vitro (Kubota et al. 2004a) and in vivo (Meng in a species where transplantation is not an option
et al. 2000). Through withdrawal and/or addition of for characterizing SSCs.
GDNF to SSC cultures, two groups have now demon- In addition to trying to drive SSCs towards their
strated that GDNF action is mediated by Src family typical biological end, there is evidence that SSCs
kinases acting through PI3 kinase/Akt-dependent are a source of pluripotent stem cells (Kanatsu-
pathways (Braydich-Stolle et al. 2007; Oatley et al. Shinohara et al. 2004a; Guan et al. 2006; Seandel
2007). In addition, microarray analysis identified et al. 2007). The ability to derive pluripotent stem
genes that are regulated by GDNF withdrawal in cells from adult tissues, with the consent of the
SSC cultures. The importance of three of these donor, may have some advantages over other
genes (Bcl6b, Erm and Lhx1) was confirmed by trans- approaches to pluripotency. However, more details
fecting SSC cultures with siRNAs specific for each are needed to understanding the genetic, epigenetic
gene. siRNA treatment caused decreased clump for- constitution of these cells, as well as their developmen-
mation in vitro and decreased colonization of tal potential.
recipient testes after transplantation (Oatley et al. Testicular tissues or testicular cell suspensions (con-
2006, 2007). taining SSCs) can be cryopreserved and may provide
Transfection of SSC cultures with siRNA, as an avenue for preservation of valuable strains or
described above, enables temporary knockdown of species. Honaramooz et al. (2002) recently demon-
the target gene. To achieve stable knockdown of a strated that testicular tissues from newborn mice,
target gene, short hairpin RNAs (shRNAs) can be pigs or goats can be grafted under the skin of
coupled with lentiviral vectors. Dann et al. (2008) immune-deficient mice and generate complete sper-
recently treated cultured SSCs with a lentiviral vector matogenesis. This approach has now been reported
Phil. Trans. R. Soc. B (2010)Downloaded from http://rstb.royalsocietypublishing.org/ on November 1, 2015
Review. Spermatogonial stem cells B. T. Phillips et al. 1673
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